|Publication number||US4511662 A|
|Application number||US 06/389,838|
|Publication date||16 Apr 1985|
|Filing date||18 Jun 1982|
|Priority date||18 Jun 1982|
|Also published as||DE3316451A1|
|Publication number||06389838, 389838, US 4511662 A, US 4511662A, US-A-4511662, US4511662 A, US4511662A|
|Inventors||Madelyn M. Baran, Dennis M. Bleile|
|Original Assignee||Bio-Rad Laboratories, Inc.|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (14), Non-Patent Citations (17), Referenced by (96), Classifications (14), Legal Events (6)|
|External Links: USPTO, USPTO Assignment, Espacenet|
1. Field of the Invention
The present invention relates generally to an assay for simultaneously determining the presence of T cells and B cells in a biological specimen, and more particularly, to an immunoassay for determining the relative amounts of T and B cells and subpopulations thereof by specifically labeling said cells with differentiable particles and counting the particles under a microscope.
In vertebrates, including man, T and B cells form the two primary lymphocyte populations. Upon stimulation with antigen, B cells differentiate into plasma cells which secrete antibodies specific to the antigen. The role of T cells is less well defined, although it is clear that they mediate cellular immune responses such as delayed hypersensitivity, immune surveillance and graft rejection. Certain T cell subpopulations also play a role in regulating the B cell response to antigens.
In normal individuals, the lymphocyte population in the peripheral blood comprises from about 70 to 80 percent T cells and from about 10 to 20 percent B cells with the remaining cells referred to as "null" cells. The determination of the relative number of T and B cells is clinically important in diagnosing a wide variety of immunologically-mediated diseases, including lymphomas, lymphocytic leukemias and various immuno-deficiency diseases, such as congenital sex-linked agammaglobulinemia. The determination is also useful in evaluating immunocompetance and mechanisms of tissue damage in autoimmune diseases, such as systemic lupus erythematosus.
The T cell and B cell populations may further be divided into subpopulations distinguished by the presence of characteristic antigens on the cell surface membrane. The primary subpopulations of T cells are the helper (TH) and suppressor (TS) cells which function in humoral immune response. The primary B cell subpopulations correspond to the classes of immunoglobulin (Ig) present on the cell surface and produced by the plasma cell after stimulation. Determination of the relative amounts of the subpopulations of both B cells and T cells also promises to have clinical importance.
It is thus useful and desirable to provide a convenient and accurate method for determining the relative amounts of T cells and B cells and subpopulations thereof in a biological sample, such as human blood.
2. Description of the Prior Art
Heretofore, human B cells have been identified based on the presence of membrane-bound immunoglobulin. Typically, labeled anti-(human immunoglobulin) antibody is used to separate or otherwise identify the B cell population. The use of polyclonal anti-(human immunoglobulin) antibody coupled to polyacrylamide beads is discussed in Jabs, et al., "Identifying Human Lymphocyte Subpopulations," Lab. Manag., (January, 1980). Such beads are available from Bio-Rad Laboratories, Richmond, Calif. Enzyme-labeled goat anti-(human immunoglobulin) antibody is described in Technical Bulletin No. 95, Sigma Chemical Company, St. Louis, Mo.
T cells have been conventionally identified by their ability to spontaneously bind with sheep red blood cells to form "rosettes" visible under an ordinary light microscope. A description of this technique is found in Hudson, et al, "Practical Immunology," Blackwell Scientific Publications, Oxford, pp. 301-302 (1980).
Commercial kits are presently available for the assay of the B cell and T cell populations in human blood. One such kit, available from Kallestad Laboratories, Inc., Austin, Tex., utilizes labeled anti-(human immunoglobulin) antibody for detecting B cells and sheep red blood cells for identifing T cells in separate steps. Another kit, identified as the "T & B Cell Reagent Set," manufactured by Hybritech, Inc., La Jolla, Calif., combines labeled anti-T cell antibody (monoclonal) and labeled anti-B cell antibody (monoclonal) for the enumeration of T and B cells using fluorescent microscopy.
The present invention provides a method and kit for the simultaneous determination of the relative amounts of B cells and T cells and subpopulations thereof in a lymphocyte population, usually derived from the blood or bone marrow.
The kit includes at least two classes of visually distinguishable labels. The first class usually comprises binding protein which is specific for B cells and which is immobilized on insoluble support particles, typically small polymeric particles in the size range from about 0.5 to 20 microns. The second class usually comprises binding protein which is specific for T cells and which is immobilized on particles which are usually, but need not be, similar to those of the first class. Either before or after immobilization of the binding proteins, at least one of the two classes of label is "marked" to allow the labels to be visually distinguished under a microscope. Typically, this is accomplished by dyeing the support particles of one or both of the classes prior to or after attachment of the binding protein.
The method is not limited to distinguishing B cells from T cells and can be extended to distinguising among the various subpopulations of B cells and T cells. In general, the method can be applied to distinguish any subpopulation which is characterized by the presence of a unique antigenic site on the cell surface membrane and for which a specific binding protein exists or can be obtained. Visually distinguishable labels can then be produced in the manner just described.
In performing the method, the lymphocyte population must first be isolated. In the case of blood lymphocytes this is usually accomplished by density gradient centrifugation. After isolation, the lymphocyte population is simultaneously exposed to both the B cell and T cell labels (including labels specific for any subpopulations) for a sufficient time to form "rosettes" with all the B cells and T cells. The rosettes may be visually distinguished under a microscope, and a count of the B cell rosettes and T cell rosettes directly indicates the relative number of B cells and T cells in the sample. Macrophages, a contaminant of the lymphocyte preparation, may be identified by their ability to ingest the polymeric beads which are part of the kit.
The present invention will be described in detail for the exemplary situation where the relative amounts of B cells and T cells are to be determined. In that case, only two labels, specific for B cells and T cells respectively, are required. It is to be understood that the present invention encompasses the method where one or more B cell or T cell subpopulations are to be simultaneously determined by providing label specific for the cells of the particular subpopulations.
The particle supports can be composed of a wide variety of materials and need not be a single material. The material must be capable of coupling the binding protein as well as a dye. A third criterion is that the material must be capable of forming small particles, in a range of from 0.5 to 20 microns, which are useful in the method of the present invention.
The materials of choice include a wide variety of polymeric materials, such as polyethylene, polypropylene, polyvinyl compounds such as polyvinylchloride, polyacrylonitrile, polyacrylate, polymethacrylate and copolymers thereof, polystyrene, nylon, polyterephthlate, cellulose and the like. The group includes naturally occuring polymers, particularly modified naturally occurring polymers and synthetic addition and condensation polymers. Also useful are cross-linked dextran particles and inorganic materials such as various glasses which may be derivatized to conjugate the binding protein as well as the dye.
Preferred are hydrolyzed cross-linked polyacrylamide particles which in the hydrolized form have a particle size of 0.5 to 20 microns, usually about 5 to 15 microns. It is desirable that the size of the particle correspond generally to the size of the lymphocytes, i.e., approximately 7 to 10 microns, preferably having a particle size distribution (90%) centered about 8 to 9 microns. Suitable beads are designated as P-6 Microbeads®, available from Bio-Rad Laboratories, Richmond, Calif. The P-6 Microbeads® should be sorted by size prior to use to obtain the desired particle size distribution.
Immobilization of the binding protein can be achieved by a variety of known techniques. Depending on the material as well as on the particular binding protein, physical absorption or adsorption could be employed. Preferably, however, the binding protein is covalently bonded to the particle. For example, carboxyl groups on the particle surface can be made reactive with the available amino groups on the binding protein. This is particularly useful when the dye is also bound to the bead through amino groups, as is the preferred technique in the present invention. Extensive literature is available concerning the binding of proteins and other molecules to surfaces employing activated carboxylic acids, carbodiimides, imido esters, active alkyl halides, etc., to form amido, amidine or amino linkages.
The preferred P-6 Microbeads® may be activated by hydrolyzing in 2N sodium hydroxide. When the beads are to be dyed, they should be hydrolyzed to a binding capacity of at least six meq (milliequivalents)/gm, typically requiring three days at room temperature. When the beads are not to be dyed, a binding capacity of 3 meq/gm is sufficient, requiring hydrolysis for only 18 to 24 hours at room temperature. The hydrolyzed P-6 Microbeads® are then equilibrated against coupling buffer prior to incubation with the binding protein and dyeing, as will now be described.
The present invention requires the immobilization of B cell binding protein (i.e., proteins capable of specifically binding to B cells) on solid phase particles, typically comprising insoluble beads as just described. B cell binding protein will usually be either monoclonal or polyclonal anti-(human immunoglobulin) antibody. Polyclonal anti-(human immunoglobulin) antibody is available from a number of sources, including Accurate Chemical and Scientific Corporation, Westbury, N.Y., [rabbit anti-(human immunoglobulin) antiserum manufactured by Dako, Denmark] and Miles Laboratories, Elkhart, Ind. [goat anti-(human immunoglobulin) antiserum]. When it is desired to further distinguish between particular B cell subpopulations, it will of course be necessary to utilize an antibody specific for the particular class of immunoglobulin, i.e., IgG, IgM, IgA, IgD and IgE, present on the B cell surface membrane.
Using the activated P-6 Microbeads® described above, antibody can be covalently bound as follows. An immunoglobulin fraction of rabbit anti-(human immunoglobulin) antiserum is dialyzed overnight in the cold against coupling buffer (e.g., 0.003M Na phosphate, pH 6.3) and a predetermined amount of immunoglobulin is then added to the activated beads and the pH adjusted in the range from 6.2 to 6.4. After thirty minutes at room temperature, EDAC [1-ethyl-3(3-dimethylaminopropyl) carbodiimide-HCl] is added and the coupling reaction is allowed to continue for about 5 to 15 hours in the cold. The amount of immunoglobulin reacted is not critical, although it is necessary that sufficient anti-Ig antibodies be coupled to the beads to assure their ability to bind B cells. In the above procedure, an immunoglobulin ooncentration of from 8 to 48 mg immunoglobulin per gram of bead has been found suitable.
The preferred T cell binding protein (i.e., protein capable of specifically binding to T cells) is monoclonal anti-(T cell) antibody, such as T101 obtained from the Murine Hybridoma Cell Line, ATCC aquisition number CRL-8023. Freshly isolated hybridoma cultures may be grown in vitro and the monoclonal antibody recovered from the supernatant fluid.
Alternatively, large amounts of hybridoma-derived antibody may be produced by injecting the tumorogenic hybridoma line into histocompatible (or immunoincompetent) mice. The procedure is well known in the art and described in Hudson, et al, Id, at pages 320-321.
Ascitic fluid obtained from the mice often contains up to 10 mg/ml of antibody, but also contains other proteins. The antibodies may be purified by affinity chromatography on immobilized protein A, such as Protein A-Sepharose® CL4B (Pharmacia Fine Chemicals, Piscataway, N.J.). An ammonium sulfate fraction of the ascitic fluid is dialyzed against buffer (pH 8.0) and is applied to the column. After washing with 0.1M phosphate buffer (pH 8.0), the monoclonal antibody is eluted with 0.1M Na citrate (pH 3.0) and dialyzed overnight in the cold against phosphate buffered saline (PBS) at pH 7.2.
The purified monoclonal antibody may be coupled to hydrolyzed P-6 Microbeads, prepared as previously described. After dialyzing against coupling buffer, the purified monoclonal antibody is added to the hydrolyzed P-6 Microbeads® at a concentration of from 5 to 40 mg antibody/gm microbeads, preferably 10 mg/gm, and pH is adjusted in the range from 6.2 to 6.4. After thirty minutes at room temperature, EDAC is added to the mixture to a final concentration in the range from 0.2 to 0.5 mg/g. The pH is adjusted in the range from 6.2 to 6.4 and the reaction is allowed to continue for a period from about 5 to 15 hours in the cold. The beads are kept in suspension, typically with an end-over-end rocker or an overhead stirring device.
After incubation, the beads are washed three times with a solution of 0.1% bovine serum albumin (BSA) in PBS, three times with a solution of 1.4M NaCl and 0.1% BSA in PBS, and three times with 0.1% BSA in PBS.
The labels must be marked in some convenient manner so that the B cell beads may be visually distinguished from the T cell beads when viewed under a microscope. Most conveniently, this is accomplished by dyeing one or both of the B cell and T labels, usually by covalently attaching a dye which is visible under a light microscope. In the past, it has been difficult to discern under a light microscope whether or not a small particle, such as the polymeric particles employed in the present invention, have been dyed. The specific procedure now described overcomes this problem.
In the preferred embodiment, the B cell labels are dyed after the particles have been conjugated to anti-(human immunoglobulin) antiserum. The method would work equally well, however, if the T cell labels were dyed. Moreover, the label particles could be dyed prior to coupling the binding protein so long as sufficient activated sites remained on the particle for coupling the binding protein. When dyeing the labels after binding protein conjugation, it is desirable to limit the concentration of dye to prevent inactivation of binding protein.
When distinguishing only B cells and T cells (and no subpopulations) it is preferable to dye only one of the two labels since it is easier to distinguish between "dyed" and "not dyed" than between two different colors. When assaying the subpopulations as well, however, it is necessary to dye more than one of the labels so that each particular label is distinguishable.
The particles may be dyed directly following conjugation of the receptor by centrifuging the beads, decanting the supernatant, and resuspending the beads in a solution of 0.2 weight percent Bismark Brown Y (see, Welcher, "Organic Analytical Reagents," Van Norstrand Co., N.Y., 1948) in coupling buffer, one volume of beads to about eight volumes of dye. After 30 minutes, EDAC (250 mg/gm beads) is added and the reaction is allowed to proceed for from 5 to 15 hours in the cold. The beads are then washed with PBS (0.1% BSA) and PBS (1.4M NaCl, 1% BSA) as set forth previously.
The above procedure employing EDAC to couple the dye to hydrolyzed beads is applicable to any dye having free amino groups. The dye will preferably have a molecular weight below 5000, usually below 1000, so that it may react with hydrolyzed groups interior to the bead and unavailable to the binding proteins.
Although the preferred embodiment utilizes labels which are dyed to be distinguishable under a visible light microscope, the invention extends to fluorescent dyes which may be visually distinguished under a fluorescent microscope, as well as to enzyme systems which generate chromogens at or near the surface of the beads, and the like.
In order to assure that sufficient labels will be available to react with every B and T cell in the sample, it is necessary that the label/cell ratio be maintained in excess, typically in the range from about 50 to 100 labels for each B and T cell. The absolute concentration of markers in the reagent solution added to the sample will, of course, depend on the sample size as well as on the concentration of B and T cells in the sample.
The method of the present invention may be practiced on any lymphocyte population isolated from a vertebrate. For example, lymphocyte suspensions may be prepared from various organs such as spleen, lymph nodes, bursa or thymus by teasing the organs apart with forceps so that cells are forced into tissue culture medium, or by teasing the organ with fine forceps over a fine wire mesh or tea strainer. Specific methods for preparing such suspensions are discussed in Hudson, et al, Id, at p. 19. The particular B cell and T cell binding proteins disclosed hereinabove are, of course, specific for the human lymphocyte population and other binding protein will be required for species other than man.
Usually, the method of the present invention will be applied to the human blood lymphocyte population which may be obtained from a blood sample. In that case, it is necessary to separate or remove the red blood cells from the blood prior to the assay. Many different methods are available for achieving such a separation. Preferred is differential centrifugation on a density gradient which is rapid and yields a high purity lymphocyte preparation.
The procedure for density gradient centrifugation is as follows. Appropriately diluted blood (3-5 ml) is layered onto a density gradient formulated such that only the red cells and granulocytes form a pellet. Suitable density gradient preparations include Ficoll-Paque® (Pharmacia Fine Chemicals, Piscataway, N.J.) and Histopaque®-1077 (Sigma Chemical Company, St. Louis, Mo.). The sample is then centrifuged at 400 g for 20 minutes at 18° to 20° C. The lymphocytes with contaminating macrophages appear at the plasma/density gradient interface. Lymphocytes are then washed from one to three times with balanced salt solution and pelleted by centrifugation at from 100 to 150 g for from 5 to 10 minutes at 18° to 20° C. The separated cells contain the lymphocytes (B cells, T cells and "null" cells) as well as macrophages, all of which are indistinguishable under a light microscope.
As an alternative to density gradient centrifugation, the red blood cells may be removed from the blood sample by first lysing the red cells in the whole blood and then washing away the lysed cells. While this method is functional, the lymphocytes are contaminated with granulocytes (another type of white blood cell). Although most granulocytes are phagocytic and do not significantly alter the T-B cell ratio, their presence decreases the absolute concentration of lymphocytes requiring a longer period to count a sufficient number of lymphocytes to provide a statistically reliable measure of the ratio.
After isolation, the lymphocyte population may be incubated with the B cell and T cell labels at a concentration (labels/cell) of 10:1 having no upper limit, preferably at least 50:1 with an upper limit of 500:1. The labels will form rosettes by binding about the periphery of the homologous cell (B or T or a particular subpopulation thereof, depending on the specificity of the label) in a manner similar to the rosettes formed by sheep red blood cells about T cells. The labels which form rosettes may then be counted under a visible light microscope, with the B cell rosettes distinguished by their darker color resulting from the dye. A count of at least 100, preferably 200, rosettes is then made to determine the ratio of B cells and T cells to the total cell population.
Incubation is carried out at room temperature for a period of at least 5 min., preferably 30 min. or longer. The longer incubation period allows macrophage ingestion of both the B cell and T cell labels. Macrophage ingestion is clearly visible under the microscope and allows differentiation of the remaining lymphocyte population ("null" cells) and the macrophages, which are otherwise indistinguishable.
B cell and T cell markers were prepared using P-6 Microbeads®, T101 monoclonal anti-(T cell) antibody and polyclonal anti-(human immunoglobulin) antibody (Dako), as described previously.
A sample of normal blood (4 ml) was diluted (1:1) with PBS, layered onto Ficoll-Paque® (3 ml), and centrifuged at 400 g for 20 min. The white cell layer was removed and washed one time with PBS (0.1% BSA) and incubated for 30 min. at 37° C. to release cytophilic Ig from the lymphocytes. After washing twice with PBS, the cell concentration was adjusted to approximately 107 cells/ml. The cells (100 μl) and labels (200 μl of both labels, each at approximately the same concentration) were placed in a test tube and incubated for 30 min. at room temperature. After centrifuging for 3 min., the cells were resuspended in PBS, 100 μl of 0.03% trypan blue in PBS was added, and one drop of the suspension placed on a microscope slide. Rosettes (200) were randomly counted and the number of B cell rosettes and T cell rosettes recorded. Only viable cells (i.e., those which excluded the trypan blue dye) were counted. The results indicated 75.8% T cells, 13.3% B cells and 7.7% null cells. These results fall well within the range of expected results. Macrophages with intracellular beads were also clearly identifiable.
The same protocol was applied to a sample of blood from a patient who was diagnosed as having chronic lymphocytic leukemia. In this case, the following results were obtained: 21% T cells, 71% B cells and 8% null cells.
Although the best mode contemplated for carrying out the present invention has been herein shown and described, it will be appreciated that variations and modifications may be made without departing from what is regarded to be the subject matter of the present invention.
|Cited Patent||Filing date||Publication date||Applicant||Title|
|US3088875 *||27 Oct 1959||7 May 1963||Hyland Lab||Immunological diagnostics utilizing polystyrene latex particles of 0.15 to 0.25 micron|
|US3853987 *||1 Sep 1971||10 Dec 1974||W Dreyer||Immunological reagent and radioimmuno assay|
|US4105598 *||3 Feb 1977||8 Aug 1978||California Institute Of Technology||Cell specific, variable density, polymer microspheres|
|US4223005 *||15 Feb 1979||16 Sep 1980||University Of Illinois Foundation||Antibody coated bacteria|
|US4254096 *||4 Oct 1979||3 Mar 1981||Bio-Rad Laboratories, Inc.||Reagent combination for solid phase immunofluorescent assay|
|US4284412 *||13 Jul 1979||18 Aug 1981||Ortho Diagnostics, Inc.||Method and apparatus for automated identification and enumeration of specified blood cell subclasses|
|US4361549 *||26 Apr 1979||30 Nov 1982||Ortho Pharmaceutical Corporation||Complement-fixing monoclonal antibody to human T cells, and methods of preparing same|
|US4361550 *||4 Dec 1979||30 Nov 1982||Ortho Pharmaceutical Corporation||Complement-fixing monoclonal antibody to human suppressor T cells and methods of preparing same|
|US4363799 *||20 Mar 1979||14 Dec 1982||Ortho Pharmaceutical Corporation||Monoclonal antibody to human T cells, and methods for preparing same|
|US4364932 *||9 Oct 1979||21 Dec 1982||Ortho Pharmaceutical Corporation||Monoclonal antibody to human cytotoxic and suppressor T cells and methods of preparing same|
|US4364937 *||8 Jan 1980||21 Dec 1982||Ortho Pharmaceutical Corporation||Monoclonal antibody to a human T cell antigen and methods of preparing same|
|US4381295 *||26 Apr 1979||26 Apr 1983||Ortho Pharmaceutical Corporation||Monoclonal antibody to human helper T cells and methods of preparing same|
|US4419444 *||30 Sep 1980||6 Dec 1983||Institut National De La Sante Et De La Recherche Medicale-Inserm||Novel supports carrying side chains, processes for obtaining these supports, process for attaching organic compounds having carbohydrate residues one said supports, products and reagents resulting from said chemical fixation|
|EP0045103A2 *||6 Jul 1981||3 Feb 1982||Akzo Nobel N.V.||Method for the determination of antigens with the aid of two or more monoclonal antibodies|
|1||*||Hudson and Hay Practical Immunology Blackwell Scientific Publications, Second Edition.|
|2||Hudson and Hay-"Practical Immunology" Blackwell Scientific Publications, Second Edition.|
|3||*||Identifying Human Lymphocyte Subpopulations and Assaying of Human Neutrophil Function (Reprints from Bio Rad Laboratories).|
|4||Identifying Human Lymphocyte Subpopulations and Assaying of Human Neutrophil Function (Reprints from Bio-Rad Laboratories).|
|5||*||Kallestad Laboratories, Inc. Kallestand Announces Development of Cellular Immunology Kits (2 pg. article).|
|6||Kallestad Laboratories, Inc.-Kallestand Announces Development of Cellular Immunology Kits (2 pg. article).|
|7||*||Mirro et al., J. Immunol. Methods, 47, 39 48, (1981).|
|8||Mirro et al., J. Immunol. Methods, 47, 39-48, (1981).|
|9||*||Parish et al., J. Immunol. Methods, 53: 367 372, (1982).|
|10||Parish et al., J. Immunol. Methods, 53: 367-372, (1982).|
|11||*||Sevier et al., Clin. Chem., 27(6), 1109, Jun. (1981).|
|12||Sigma Technical Bulletin #95, "An Immunoenzymatic Technic for Detecting Surface Immunoglobulins of Mature B-Lymphocytes, (Nov. 1980).|
|13||*||Sigma Technical Bulletin 95, An Immunoenzymatic Technic for Detecting Surface Immunoglobulins of Mature B Lymphocytes, (Nov. 1980).|
|14||*||T & B Lymphocyte Identification with Monoclonal Antibodies Hybritech T & B Cell Reagent Set from Hybritech Inc., (1981), (4 pgs.).|
|15||T & B Lymphocyte Identification with Monoclonal Antibodies-Hybritech T & B Cell Reagent Set from Hybritech Inc., (1981), (4 pgs.).|
|16||*||The First Lymphochrome B Lymphocyte Test (Sigma Chemical Corp., 1 pg. brochure), (1980).|
|17||The First Lymphochrome™ B-Lymphocyte Test (Sigma Chemical Corp., 1 pg. brochure), (1980).|
|Citing Patent||Filing date||Publication date||Applicant||Title|
|US4584277 *||5 Apr 1983||22 Apr 1986||Syntex (U.S.A.) Inc.||Fluorescent multiparameter particle analysis|
|US4649106 *||1 Jun 1984||10 Mar 1987||Dana-Farber Cancer Institute, Inc.||Distinguishing subsets of human cells|
|US4657852 *||4 Jun 1984||14 Apr 1987||The Board Of Trustees Of The Leland Stanford Junior University||Method for B cell typing of total human lymphocyte sample|
|US4713348 *||22 Oct 1985||15 Dec 1987||Syntex (U.S.A.) Inc.||Fluorescent multiparameter particle analysis|
|US4716107 *||10 Dec 1986||29 Dec 1987||Gross Robert L||Method for diagnosis of A.I.D.S.|
|US4745075 *||26 Aug 1985||17 May 1988||Burroughs Wellcome Co.||Diagnostic test methods|
|US4797363 *||14 Jul 1986||10 Jan 1989||Board Of Trustees, University Of Illinois||Bacteriophages as recognition and identification agents|
|US4828984 *||11 Apr 1986||9 May 1989||Flow Cytometry Standards Corporation||Composition, synthesis and use of simulated cells|
|US4960713 *||25 Feb 1988||2 Oct 1990||Burroughs Wellcome Co.||Diagnostic test methods|
|US4960714 *||25 Feb 1988||2 Oct 1990||Burroughs Wellcome Co.||Diagnostic test methods|
|US4960715 *||25 Feb 1988||2 Oct 1990||Burroughs Wellcome Co.||Diagnostic test methods|
|US5030561 *||27 Dec 1988||9 Jul 1991||Becton, Dickinson And Company||Chlamydia assay using amidine modified supports or particles|
|US5071774 *||23 Jun 1988||10 Dec 1991||Syntex (U.S.A.) Inc.||Multiparameter particle analysis|
|US5073497 *||16 Jan 1990||17 Dec 1991||Caribbean Microparticles Corporation||Microbead reference standard and method of adjusting a flow cytometer to obtain reproducible results using the microbeads|
|US5073498 *||16 Mar 1990||17 Dec 1991||Caribbean Microparticles Corporation||Fluorescent alignment microbeads with broad excitation and emission spectra and its use|
|US5084394 *||17 Apr 1990||28 Jan 1992||Vogt Robert F||Method for corrective calibration of a flow cytometry using a mixture of fluorescent microbeads and cells|
|US5089416 *||21 Nov 1990||18 Feb 1992||Caribbean Microparticles Corporation||Method of use of non-fluorescent particles to determine fluorescence threshold of a flow cytometer relative to the autofluorescence of samples|
|US5116724 *||29 Sep 1988||26 May 1992||Immunotech||Products for separation applicable to cells in the immunopurification field|
|US5238812 *||1 May 1990||24 Aug 1993||Coulter Corporation||Method and apparatus for rapid mixing of small volumes for enhancing biological reactions|
|US5246829 *||10 Mar 1992||21 Sep 1993||Immunotech||Products for separation applicable to cells in the immunopurification field|
|US5314824 *||27 Oct 1992||24 May 1994||Caribbean Microparticles Corporation||Method of setting up a flow cytometer|
|US5340719 *||23 Nov 1990||23 Aug 1994||Corporation Coulter||Method and apparatus for optically screening microscopic cells|
|US5348859 *||20 May 1991||20 Sep 1994||Coulter Corporation||Method and apparatus for obtaining an absolute white blood cell subset count and white blood cell multipart differential|
|US5380663 *||23 Aug 1993||10 Jan 1995||Caribbean Microparticles Corporation||Automated system for performance analysis and fluorescence quantitation of samples|
|US5468616 *||29 Apr 1993||21 Nov 1995||Coulter Corporation||Method and apparatus for rapid mixing of small volumes for enhancing biological reactions|
|US5554505 *||1 Dec 1993||10 Sep 1996||Coulter Corporation||Method and apparatus for optically screening microscopic cells|
|US5561045 *||4 Jan 1994||1 Oct 1996||Intracel Corporation||Detection reagent, article, and immunoassay method|
|US5646004 *||31 Aug 1994||8 Jul 1997||Activated Cell Therapy, Inc.||Methods for enriching fetal cells from maternal body fluids|
|US5648223 *||31 Aug 1994||15 Jul 1997||Activated Cell Therapy, Inc.||Methods for enriching breast tumor cells|
|US5663051 *||11 Dec 1995||2 Sep 1997||Activated Cell Therapy, Inc.||Separation apparatus and method|
|US5840502 *||31 Aug 1994||24 Nov 1998||Activated Cell Therapy, Inc.||Methods for enriching specific cell-types by density gradient centrifugation|
|US6893881||10 Sep 1993||17 May 2005||Abbott Laboratories, Inc.||Method for detection of specific target cells in specialized or mixed cell population and solutions containing mixed cell populations|
|US7179391||23 May 2003||20 Feb 2007||Biomet Manufacturing Corp.||Apparatus and method for separating and concentrating fluids containing multiple components|
|US7374678||2 Sep 2004||20 May 2008||Biomet Biologics, Inc.||Apparatus and method for separating and concentrating fluids containing multiple components|
|US7470371||19 Oct 2006||30 Dec 2008||Hanuman Llc||Methods and apparatus for isolating platelets from blood|
|US7771590||23 Aug 2005||10 Aug 2010||Biomet Manufacturing Corp.||Method and apparatus for collecting biological materials|
|US7780860||19 May 2008||24 Aug 2010||Biomet Biologics, Llc||Apparatus and method for separating and concentrating fluids containing multiple components|
|US7806276||11 Apr 2008||5 Oct 2010||Hanuman, Llc||Buoy suspension fractionation system|
|US7832566||25 May 2006||16 Nov 2010||Biomet Biologics, Llc||Method and apparatus for separating and concentrating a component from a multi-component material including macroparticles|
|US7837884||29 Dec 2008||23 Nov 2010||Hanuman, Llc||Methods and apparatus for isolating platelets from blood|
|US7845499||25 May 2006||7 Dec 2010||Biomet Biologics, Llc||Apparatus and method for separating and concentrating fluids containing multiple components|
|US7914689||19 May 2008||29 Mar 2011||Biomet Biologics, Llc||Apparatus and method for separating and concentrating fluids containing multiple components|
|US7992725||11 Apr 2008||9 Aug 2011||Biomet Biologics, Llc||Buoy suspension fractionation system|
|US8048297||3 May 2007||1 Nov 2011||Biomet Biologics, Llc||Method and apparatus for collecting biological materials|
|US8048320||17 Jun 2010||1 Nov 2011||Biomet Manufacturing Corp.||Method and apparatus for collecting biological materials|
|US8048321||11 Aug 2010||1 Nov 2011||Biomet Biologics, Llc||Apparatus and method for separating and concentrating fluids containing multiple components|
|US8062534||6 Dec 2010||22 Nov 2011||Biomet Biologics, Llc||Apparatus and method for separating and concentrating fluids containing multiple components|
|US8097429 *||1 Feb 2005||17 Jan 2012||Fodstad Oeystein||Method for characterization of abnormal cells|
|US8119013||4 Oct 2010||21 Feb 2012||Hanuman, Llc||Method of separating a selected component from a multiple component material|
|US8163184||25 Mar 2011||24 Apr 2012||Biomet Biologics, Llc||Apparatus and method for separating and concentrating fluids containing multiple components|
|US8187477||22 Nov 2010||29 May 2012||Hanuman, Llc||Methods and apparatus for isolating platelets from blood|
|US8236258||31 Oct 2011||7 Aug 2012||Biomet Biologics, Llc||Method and apparatus for collecting biological materials|
|US8313954||3 Apr 2009||20 Nov 2012||Biomet Biologics, Llc||All-in-one means of separating blood components|
|US8328024||4 Aug 2011||11 Dec 2012||Hanuman, Llc||Buoy suspension fractionation system|
|US8337711||27 Feb 2009||25 Dec 2012||Biomet Biologics, Llc||System and process for separating a material|
|US8512575||6 Aug 2012||20 Aug 2013||Biomet Biologics, Llc||Method and apparatus for collecting biological materials|
|US8567609||19 Apr 2011||29 Oct 2013||Biomet Biologics, Llc||Apparatus and method for separating and concentrating fluids containing multiple components|
|US8591391||12 Apr 2010||26 Nov 2013||Biomet Biologics, Llc||Method and apparatus for separating a material|
|US8596470||20 Feb 2012||3 Dec 2013||Hanuman, Llc||Buoy fractionation system|
|US8603346||22 Sep 2011||10 Dec 2013||Biomet Biologics, Llc||Apparatus and method for separating and concentrating fluids containing multiple components|
|US8783470||25 May 2012||22 Jul 2014||Biomet Biologics, Llc||Method and apparatus for producing autologous thrombin|
|US8801586 *||20 Dec 2012||12 Aug 2014||Biomet Biologics, Llc||System and process for separating a material|
|US8808551||15 Nov 2010||19 Aug 2014||Biomet Biologics, Llc||Apparatus and method for separating and concentrating fluids containing multiple components|
|US8950586||1 Jul 2013||10 Feb 2015||Hanuman Llc||Methods and apparatus for isolating platelets from blood|
|US8992862||15 Nov 2012||31 Mar 2015||Biomet Biologics, Llc||All-in-one means of separating blood components|
|US9011800||16 Jul 2009||21 Apr 2015||Biomet Biologics, Llc||Method and apparatus for separating biological materials|
|US9114334||9 Dec 2013||25 Aug 2015||Biomet Biologics, Llc||Apparatus and method for separating and concentrating fluids containing multiple components|
|US9138664||2 Dec 2013||22 Sep 2015||Biomet Biologics, Llc||Buoy fractionation system|
|US9239276||28 Oct 2013||19 Jan 2016||Biomet Biologics, Llc||Apparatus and method for separating and concentrating fluids containing multiple components|
|US9533090||21 Nov 2013||3 Jan 2017||Biomet Biologics, Llc||Method and apparatus for separating a material|
|US9556243||10 Oct 2013||31 Jan 2017||Biomet Biologies, LLC||Methods for making cytokine compositions from tissues using non-centrifugal methods|
|US9642956||27 Aug 2012||9 May 2017||Biomet Biologics, Llc||Apparatus and method for separating and concentrating fluids containing multiple components|
|US9649579||10 Dec 2012||16 May 2017||Hanuman Llc||Buoy suspension fractionation system|
|US9701728||24 Jul 2015||11 Jul 2017||Biomet Biologics, Llc||Methods and compositions for delivering interleukin-1 receptor antagonist|
|US9719063||11 Aug 2014||1 Aug 2017||Biomet Biologics, Llc||System and process for separating a material|
|US20050109716 *||2 Sep 2004||26 May 2005||Michael Leach||Apparatus and method for separating and concentrating fluids containing multiple components|
|US20050130234 *||1 Feb 2005||16 Jun 2005||Oystein Fodstad||Method for characterization of abnormal cells|
|US20060273049 *||25 May 2006||7 Dec 2006||Leach Michael D||Method and apparatus for separating and concentrating a component from a multi-component material including macroparticles|
|US20060273050 *||25 May 2006||7 Dec 2006||Higgins Joel C||Apparatus and method for separating and concentrating fluids containing multiple components|
|US20060278588 *||26 May 2006||14 Dec 2006||Woodell-May Jennifer E||Apparatus and method for separating and concentrating fluids containing multiple components|
|US20070075016 *||23 Aug 2005||5 Apr 2007||Biomet Manufacturing Corp.||Method and apparatus for collecting biological materials|
|US20070208321 *||3 May 2007||6 Sep 2007||Biomet Manufacturing Corp.||Method And Apparatus For Collecting Biological Materials|
|US20080217264 *||19 May 2008||11 Sep 2008||Biomet Manufacturing Corp.||Apparatus And Method For Separating And Concentrating Fluids Containing Multiple Components|
|US20080217265 *||19 May 2008||11 Sep 2008||Biomet Manufacturing Corp.||Apparatus And Method for Separating And Concentrating Fluids Containing Multiple Components|
|US20080283474 *||11 Apr 2008||20 Nov 2008||Biomet Biologics, Llc||Buoy suspension fractionation system|
|US20100255977 *||17 Jun 2010||7 Oct 2010||Biomet Manufacturing Corp.||Method and Apparatus for Collecting Biological Materials|
|US20130196425 *||20 Dec 2012||1 Aug 2013||Biomet Biologics, Llc||System and Process for Separating a Material|
|USRE43979||5 Feb 2003||5 Feb 2013||Abbott Laboratories||Method for detection of specific target cells in specialized or mixed cell population and solutions containing mixed cell populations|
|EP1151298A1 *||13 Jan 2000||7 Nov 2001||Biocrystal Limited||Methods and assay kits for detecting mononuclear cell phenotype|
|EP1151298A4 *||13 Jan 2000||24 Oct 2002||Biocrystal Ltd||Methods and assay kits for detecting mononuclear cell phenotype|
|WO1985004189A1 *||15 Mar 1985||26 Sep 1985||Teodorescu Marius C||Bacteriophages as recognition and identification agents|
|WO1985005639A1 *||10 May 1985||19 Dec 1985||Dana-Farber Cancer Institute, Inc.||Distinguishing subsets of human cells|
|WO1988006918A1 *||11 Mar 1988||22 Sep 1988||Coulter Electronics, Inc.||Method and apparatus for rapid mixing of small volumes for enhancing biological reactions|
|WO1992009878A1 *||22 Nov 1991||11 Jun 1992||Coulter Corporation||Method and apparatus for optically screening microscopic cells|
|WO2017124101A2||17 Jan 2017||20 Jul 2017||The Broad Institute Inc.||Semi-permeable arrays for analyzing biological systems and methods of using same|
|WO2017136751A1||3 Feb 2017||10 Aug 2017||The Broad Institute Inc.||Multi-stage, multiplexed target isolation and processing from heterogeneous populations|
|U.S. Classification||436/513, 436/548, 436/533, 436/519, 436/531, 436/523, 436/534|
|International Classification||G01N33/53, A61K35/26, G01N33/569|
|Cooperative Classification||A61K35/26, G01N33/56972|
|European Classification||G01N33/569H2, A61K35/26|
|21 Oct 1982||AS||Assignment|
Owner name: BIO-RAD LABORATORIES, INC. 2200 WRIGHT AVE. RICHMO
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNORS:BARAN, MADELYN M.;BLEILE, DENNIS M.;REEL/FRAME:004055/0008
Effective date: 19820614
|23 May 1988||FPAY||Fee payment|
Year of fee payment: 4
|8 Jun 1992||FPAY||Fee payment|
Year of fee payment: 8
|19 Nov 1996||REMI||Maintenance fee reminder mailed|
|13 Apr 1997||LAPS||Lapse for failure to pay maintenance fees|
|24 Jun 1997||FP||Expired due to failure to pay maintenance fee|
Effective date: 19970416